This project aims to evaluate microscale methods for use in studies of pollutant bioavailability in soil and sediment. The overall goal is to provide a better mechanistic understanding of microbiological and microenvironmental favors controlling the distribution, biodegradation and transport of pollutants such as mono- and polycyclic aromatic hydrocarbons (PAHs) (e.g. toluene, naphthalene and phenanthrene) and heavy metals (e.g. Cd and Pb) in porous media. The approach is to develop and apply microscopic detection and identification methods such as fluorescent antibody (Fab) staining, microautoradiography (MARG), and oligonucleotide fluorescent in situ hybridization (FISH) to determine the microscale distribution and differential activity patterns of specific bacteria (and genes) and pollutant chemicals relative to non-living components of soil and sediment. Conventional light and electron microscopic methods will also be used for differential staining and identification of the non-living components. Application of these microscale methods will result in a better understanding of pollutant distribution and availability relative to the non-living components and the bacterial cells capable that participate in their biodegradation or transport. In the continuation project, a newly developed Fab-MARG procedure will be refined in combined macro- and microscale studies of toluene biodegradation by Pseudomonas putida Fl in organic sediment and subsequently be applied in similar studies with PAHs. New FISH-MARG methods will be developed for identifying non-culturable bacteria using a fluorescently labeled ribosomal RNA-targeted oligonucleotide hybridization variation of the Fab-MARG procedure. Fab detection methods will be tested for identifying pollutant chemicals and humic components in soil or sediment matrix. Other differential light and electron microscopic methods, including STEM-MARG and STEM-EDX, autofluorescence and fluorochrome staining of soil matrix components, and colloidal gold- labeled antibody staining, will also be tested for their efficacy in estimating pollutant distribution in relation to the microstructure of soil and sediment particles. A macroscale model structure was developed with microscale model components embedded in it that allow for direct application of microscale data to macroscale performance. Biodegradation kinetic and pollutant distribution parameters for use in this structured model will be evaluated using the microscale methodologies as they become available to confirm the interrelationships between the physical and chemical availability of the contaminants and their mineralization rates in soils and sediments. The mechanistic understanding obtained in the proposed research will provide the basis for continued development of more robust models for pollutant bioavailability, degradation, and transport than have been available before. Development of such models is expected to aid exposure assessment and risk management activities and reduce the costs of bioremediation by allowing for more accurate predictions of pollutant behavior than is possible with current models.